Insulin dependent diabetes mellitus (DDM) is an autoimmune disease in which there is a characteristic immunological reactivity to a limited set of tissue-specific cytoplasmic autoantigens of pancreatic islet beta cells. Reactivity to one of these autoantigens, glutamic acid decarboxylase (GAD; EC 4.1.1.15), is virtually unique to the disease, the rare exceptions being the neurological disorder, Stiff man syndrome (Solimena et al., 1990; Baekkeskov et al., 1990), and the polyendocrine syndrome Types 1 and 2. Glutamic acid decarboxylase catalyses the conversion of L-glutamic acid to .gamma.-aminobutyric acid (GABA) and carbon dioxide (Erlander et al., 1991). GABA is a major inhibitory neurotransmitter, and hence most research on GAD until recently had concentrated on the role of this enzyme in neural functioning. A new direction developed with the recognition that antibodies to GAD are prevalent in IDDM (Baekkeskov et al., 1990).
GAD exists as 2 isoforms denoted by their calculated molecular weights as GAD65 and GAD67. These differ according to their subcellular location (Erdo and Wolff, 1990; Faulkner-Jones et al., 1993), chromosomal location (Erlander et al. 1991; Karlsen et al., 1991; Bu et al., 1992; Michelsen et al., 1991), amino acid sequence (Bu et al., 1992) and cofactor interactions (Erlander and Tobin, 1991) but have close homology, in man 65% identity and 80% similarity. The greatest divergence between the isoforms occurs in the first 100 amino acids (Bu et al. 1992). The availability of cDNA clones encoding the two GAD isoforms has allowed the expression of these in various systems, including bacteria (Kaufman et al., 1992), Sf9 insect cells using the baculovirus vector (Seissler et al., 1993; Mauch et al., 1993), COS7 monkey cells (Velloso et al., 1993), baby hamster kidney cells (Hagopian et al., 1993), yeast (Powell et al, 1995) and by in vitro translation using rabbit reticulocyte lysate (RRL) (Petersen et al., 1994; Ujihara et al., 1994; Grubin et al., 1994). In addition, a modified GAD65 without the hydrophobic amino acids 245 inclusive of the N-terminal region has been expressed in yeast (Powell, et al., 1996).
The identification of GAD as a major autoantigen of IDDM has led to the extensive use of this antigen in immunoassays for the accurate diagnosis and prediction of IDDM in at-risk populations. Such studies have shown that antibodies to GAD are detectable in patients up to 10 years before the early onset of clinical symptoms (Baekkeskov et al., 1987, Atkinson et al., 1990; Rowley et al., 1992; Chen et al., 1993; Tuomilehto et al., 1994; Myers et al., 1996). These assays have employed autoantigenic GAD derived from two major sources. One source is animal materials, most commonly porcine brain, purified by affinity chromatography, and labelled with radioactive iodine. The other source is in vitro transcription and translation of the cloned human GAD65 gene, using rabbit reticulocyte lysate (RRL) which produces biosynthetically labelled GAD suitable for radioimmunoprecipitation (RIP) assays (Guazzaroti et al., 1995). Expression from RRL has been widely used in diagnostic assays for anti-GAD in human sera but the in vitro expression system has limitations in that only very small amounts, in the order of picomoles, of GAD are produced and the process is very costly. Bacterial expression does not appear to yield GAD that is amenable to use in diagnostic assays, and yields from mammalian cells are unsuitably low. Several authors have presented evidence that the GAD must be in a particular conformation to be reactive with antibodies in IDDM since IDDM sera generally do not show reactivity with GAD in Western blotting under denaturing conditions, yet show potent reactivity under non-denaturing conditions (Rowley et al., 1992; Tuomi et al., 1994, Myers et al., 1996); the GAD conformation that is recognised by the majority of IDDM sera is sensitive to exposure to reducing agents such as .beta.-mercaptoethanol, since GAD that has been thus treated loses reactivity (Tuomi et al., 1994). It is also recognised that antibodies to GAD65 in IDDM react with particular epitopes on the molecule that lie in the mid-region and C-terminal region of the molecule. Fusions of cDNAs that encode particular sequences of GAD65 and GAD67 have been created as "chimeric" proteins to establish epitope recognition (Daw and Powers, 1995).
Assays using immunoprecipitation and either affinity purified porcine brain GAD (Rowley et al., 1992) or recombinant GAD (Kaufman et al., 1992; Seissler et al., 1993; Mauch et al., 1993; Velloso et al., 1993; Hagopian et al., 1993; Petersen et al., 1994; Ujihara et al., 1994; Grubin et al., 1994) have revealed that 70-80% of IDDM sera contain autoantibodies to GAD65, whereas only 8-25 % contain antibodies to GAD67. The autoantibodies in Stiff-man syndrome react with GAD by immunoblotting under reducing conditions (Solimena et al., 1990), whereas autoantibodies to GAD in IDDM seldom do so (Baekkeskov et al., 1990), and are thought to recognise a conformational epitope (Tuomi et al., 1994). Epitopes have been mapped by examining the reactivity by immunoprecipitation of IDDM sera against truncated polypeptides of GAD65, with one major IDDM associated epitope located to the middle and carboxy terminal domains of GAD65 (Kaufman et al., 1992; Richter et al., 1993; Ujihara et al., 1994). More recent mapping for epitopes for anti-GAD suggests the presence of two discontinuous epitopes, one within amino acids 244 to 433, and the other within amino acids 451 to 570 (Daw and Powers, 1995). Curiously, whilst antigenically active regions of GAD65 and GAD67 are highly homologous, GAD65 is the isoform with which autoantibodies are predominantly reactive.
The further development of diagnostic assays for IDDM would greatly benefit from a more accessible source of large amounts of recombinant GAD that would be free of the possible biohazards associated with mammalian sources. Furthermore, the search for disease-associated epitopes of GAD would be facilitated by the availability of a simple system with which to carry out site-specific mutagenesis and deletion studies. The requirement for GAD to be in a particular conformation has hindered prior efforts to use recombinant DNA technology to produce large quantities of antigenically active GAD. Although the use of E. coli expression systems is reported to produce enzymatically active GAD (Kaufman et al., 1992), published reports on the utility of bacterially-expressed antigenically active GAD for immunoassays are scarce and, as stated above, it is the experience of the present inventors that this material performs less effectively in radioimmunoprecipitation tests with IDDM sera than does GAD expressed in other systems. The preferred expression system should be capable of producing not only large amounts of GAD, but also GAD that is in an appropriate conformation to be recognised by IDDM sera.